Sodium dichloroacetate improves migration ability by suppressing LPS-induced inflammation in HTR-8/SVneo cells via the TLR4/NF-κB pathway

Objective(s): Inadequate cytotrophoblast migration and invasion are speculated to result in preeclampsia, which is a pro-inflammatory condition. Sodium dichloroacetate (DCA) exerts anti-inflammatory actions. Thus,we sought to investigate the effect of DCA on the migration function of the lipopolysaccharide (LPS)-stimulated human-trophoblast-derived cell line (HTR-8/SVneo). Materials and Methods: HTR-8/SVneo cells were treated with LPS to suppress cell migration. Cell migration was examined by both scratch wound healing assay and transwell migration assay. Western blotting was used to analyze the expression levels of toll-like receptor-4 (TLR4), nuclear factor-κB (NF-κB), TNF-α, IL-1β, and IL-6 in the cells. Results: DCA reversed LPS-induced inhibition of migration in HTR-8/SVneo cells. Furthermore, DCA significantly suppressed LPS-induced activation of TLR4, phosphorylation of NF-κB (p65), translocation of p65 into the nucleus, and the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Treatment with inhibitors of TLR4 signal transduction (CLI095 or MD2-TLR-4-IN-1) reduced LPS-induced overexpression of pro-inflammatory cytokines, and a synergistic effect was found between TLR4 inhibitors and DCA in HTR-8/SVneo cells. Conclusion: DCA improved trophoblast cell migration function by suppressing LPS-induced inflammation, at least in part, via the TLR4/NF-κB signaling pathway. This result indicates that DCA might be a potential therapeutic candidate for human pregnancy-related complications associated with trophoblast disorder.


Introduction
Preeclampsia (PE) is a complex pregnancy disorder that affects 2% to 8% of pregnancies worldwide.Maternal spiral artery remodeling and poor placental implantation are associated with this condition (1-3).During pregnancy, one of the prominent factors for proper implantation and placentation is migration of extravillous trophoblast (EVT) cells into spiral arterioles.Only after invasion can trophoblast cells successfully degrade and migrate through the extracellular matrix to interact closely with the endothelial cells of the uterine spiral arteries and further replace them (4,5).Insufficient trophoblastic invasion of the decidua and spiral arteries is considered to be the first stage of PE development (6,7).For researchers in reproductive medicine, the question of how to promote trophoblast cell function is of particular interest.
Though the pathophysiology of PE remains ill-defined, there is a large body of evidence that shows the inflammatory reaction is significantly enhanced in the pathophysiology of PE, involving several pro-inflammatory factors.
Migration and invasion of trophoblast cells are affected by those cytokines (8)(9)(10)(11)(12).As a treatment for congenital lactic acidosis and other diseases, sodium dichloroacetate (DCA) is a potent and safe agent (13,14).Recent studies have found that DCA may exert some anti-inflammatory actions (15)(16)(17)(18).A study in the rheumatoid arthritis mouse model has shown that DCA administration alleviates the development of arthritis in female mice (17).Thus, it can be logically speculated that DCA could modulate trophoblast cell migration function by inhibiting the inflammatory response.
We performed lipopolysaccharide (LPS) treatment on trophoblast cells to suppress cell migration ability in this investigation.Toll-like receptor-4 (TLR4) can be activated by LPS, and TLR4 activation can recruit NF-κB and increase several chemokines and inflammatory cytokines synthesis, for example, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) (19,20).As a family member of pathogen-related molecule pattern recognition molecules, TLR4 has an integral part in the initiation and acceleration of inflammation (21).As a downstream of TLR4-mediated signaling, the NF-κB signaling pathway has a critical effect in amplifying the inflammatory response by increasing the expression of diverse pro-inflammatory cytokine genes (22).Therefore, we aimed to investigate in this study whether DCA could ameliorate LPS-induced HTR-8/SVneo cell migration ability impairment and examine if DCA showed antiinflammatory roles through suppression of the TLR4/NF-κB pathway.

Cell culture
The International Peace Maternity & Child Health Hospital of the China Welfare Institute (China) provided human chorionic trophoblast cells (HTR-8/SVneo).Cells were plated at 5 × 10 5 cells/well on a 6-well plate and cultured overnight in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) under 5% CO 2 at 37 °C (23).

Cell viability assay
The viability of cells after treatment with different doses was determined using the Cell Counting Kit-8 (Dojindo, Japan) to detect the cytotoxicity of LPS.In 96-well plates, 5000 cells per well were cultivated for 24 to 48 hr.LPS were added to different groups at 0.1, 0.2, 0.5, or 1 μg/ml final concentrations when 90% confluence had been reached.A replacement medium containing 10% CCK-8 was added after 24 hr and incubated for an additional hour.With a microplate reader, we measured optical density at 450 nm (24).

LPS treatment
Cells were cultured using HTR-8/SVneo cells according to Section 2.2, using LPS to suppress cell migration.After treating the cells with different concentrations of LPS (0.1, 0.2, 0.5, or 1 µg/ml) for 24 hr, the culture medium was changed to RPMI 1640 with 1% (v/v) FBS.We found that LPS (0.1, 0.2, 0.5, or 1 µg/ml) significantly inhibited HTR-8/SVneo cell migration.What is more, LPS-induced cell migration dysfunction was dose-dependent, and reached maximal values at 0.5 µg/ml (Figure 1b).Taking together our results as well as other researchers' study (19), we selected the LPS concentration (0.5 µg/ml) for the continuation of this study.

Scratch wound healing assay
Cell migration capability was assessed using a scratch wound healing assay.That is, cells were cultured in 6-well plates at a density of 1.0 × 10 5 , and then monolayers of cells were scraped with a pipette tip and washed with PBS.The cells were subjected to treatment conditions as stated in Sections 2.4 and 2.5.Photographs were captured utilizing an inverted microscope at two distinct time points, namely 0 hr and 24 hr.Measurements were taken at various locations to determine the width of the scratch (25,26).It was shown that DCA (0.01, 0.05, or 0.25 mmol/l) significantly improve HTR-8/SVneo cell migration when stimulated by LPS, and the effect of DCA was comparable with that of ASP (10 μg/ ml) at the concentration of 0.05 mmol/l (Figure 2a).Thus, the concentration of DCA (0.05 mmol/l) was used in the following experiments of the present work.

Transwell migration assay
By using the transwell migration assay, this assay assessed the migration potential of HTR-8/SVneo cells.A total of four different cell treatments were employed: vehicle, LPS (0.5 µg/ml), LPS (0.5 µg/ml) + DCA (0.05 mmol/l), and LPS (0.5 µg/ml l) + ASP (10 µg/ml) for 24 hr, following trypsinization, the target concentration of the cell suspension was 1.0 × 10 5 cells/ml.100 µl of serum-free medium was added to the supra-chamber and 10% FBS medium was added to the infra-chamber.Incubation for 24 hr was followed by staining with Hoechst 33258 for 15 min and fixing with 2% (v/v) paraformaldehyde.Five fields of each sample (magnification × 100) were counted randomly for the number of migrated cells, and the average of the five different regions was calculated (27)(28)(29).In all experiments, blinding and randomization were applied.
RNA isolation and cDNA preparation RNA was extracted from cells using the RNeasy Mini Kit (Qiagen).Prior to use, optical density was tested at 260 and 280 nm to define the concentration and purity of the RNA.Each sample's total RNA (1 g) was used for reverse transcription.DNA synthesized with FastKing RT kit and gDNase (Tianjian).

Inhibitor treatment
To further examine whether DCA exerted antiinflammatory roles by inhibiting the TLR4/NF-κB pathways, antibodies against TLR4 intracellular binding domain (CLI-095) and extracellular binding domain (MD2-TLR4-IN-1) were added at final concentrations of 10 µmol/L.Briefly, HTR-8/SVneo cells were pretreated with or without CLI-095 or MD2-TLR-4-IN-1 for 1 hr before incubation with LPS or DCA for 24 hr (33,34).Then, the cells were harvested by trypsin digestion and centrifugation for western blotting analysis.

Statistical analysis
The data were shown as mean ± SEM.The statistical significance of group differences was ascertained using oneway ANOVA, and a Turkey post hoc analysis was then carried out.P-values < 0.05 were regarded as significant in statistics.

Different concentrations of LPS inhibit HTR-8/SVneo cell migration
To explore how LPS affects the migration of HTR-8/SVneo cells and avoids their cytotoxicity, we conducted cell viability assays and scratch wound healing assays.Following a 24 hr treatment with different concentrations of LPS (0.1, 0.2, 0.5, or 1 μg/ml), it was discovered that cell viability remained unaffected (Figure 1a), Based on the outcomes of the cell viability experiments, the scratch assay was used to assess how different LPS dosages affected the migratory function of HTR-8/SVneo cells.It was found that LPS-induced cell migration dysfunction was dose-dependent and reached maximal values at 0.5 µg/ml (*P<0.05,**P<0.01; Figure 1b).
On the basis of these data and other researchers' study (19), the 0.5 µg/ml dose was therefore selected for subsequent experiments.

DCA improves LPS-inhibited HTR-8/SVneo cell migration
Scratch wound healing assay and transwell migration assays were used to analyze the impact of DCA on the migration function of LPS-stimulated HTR-8/SVneo cells.It was found that DCA could significantly improve cell migration distance (**P<0.01; Figure 2a) and migration number (**P<0.01; Figure 2b).The effect of DCA was comparable with that of ASP (10 μg/ml) in promoting HTR-8/SVneo cell migration at the concentration of 0.05 mmol/l.Thus, the concentration of DCA (0.05 mmol/l) was used in the following experiments of the present work.

DCA suppressed LPS-induced phosphorylation of p65 and the translocation of p65 into the nucleus
It has been suggested that NF-κB played a crucial role in the regulation of pro-inflammatory cytokine production.Downstream of the NF-κB pathway is activated by TLR4 signaling.Previous investigations by others have shown that activation of the NF-κB pathway leads to results in translocation of NF-κB subunits including phospho-p65 to the nucleus (35)(36)(37).We investigated whether the NF-κB signaling pathway was responsible for DCA's ability to reduce inflammatory responses in LPS-stimulated HTR-8/SVneo cells.HTR-8/SVneo cells were co-treated with DCA (0.05 mmol/l) and LPS (0.5 µg/ml) for 24 hr, then the expression levels of phosphorylation of p65 and p65 were detected and examined using blotting.As with cells treated with ASP, we discovered that DCA (0.05 mmol/L) dramatically reduced the phosphorylation of p65 when compared to the control (**P<0.01; Figure 5a-d).We also examined whether the translocation of the p65 subunit of NF-κB from the cell membrane to the nucleus could be disrupted by DCA.NF-κB p65 staining in the nucleus indicated that most intracellular p65 translocated from the cytoplasm to the nucleus in LPS-stimulated cells.By treating cells with DCA (0.5 μg/ml), there was a significant reduction in p65 levels in the nucleus (Figure 5e).

Discussion
In the present study, we found that DCA promoted the migration of LPS-stimulated HTR-8/SVneo cells by suppressing inflammatory response via the TLR4/NF-κB pathway, and the promoting effect of DCA was comparable with that achieved with aspirin (an agent might prevent or delay pre-eclampsia in randomized trials) (38).
The significance of the epithelial-mesenchymal transition (EMT) during the differentiation of extravillous cytotrophoblasts (evCTB) is well established (39).Epithelial cells undergo a cellular process called EMT that transforms them into mesenchymal cells, enabling them to migrate and metastasize (39).EMT has proved to play a major role in both early and late development of trophoblasts (40).The HTR-8/SVneo cell line is a heterogeneous population of trophoblast and stromal cells (41).As a result of further investigations, it was identified that the heterogeneity of the cells was mainly attributed to the ongoing EMT process in   vitro, which resembles the EMT that occurs during normal trophoblast development (40).Moreover, across different placental cell lines, HTR-8/SVneo is still the one that has been most frequently used to study the invasion, migration, and proliferation of evCTB, a cell line that develops from first-trimester evCTB infected with retroviral vectors (40).
Clinically, DCA is used to treat lactic academia, including mitochondrial encephalomyopathy and pyruvate dehydrogenase complex deficiency (13).There have been 40 years of human experience with mechanistic investigations of DCA in human tissues following oral administration, and it has been demonstrated that DCA is powerful, longlasting, and safe in both children and adults (14).The present study demonstrated that DCA could significantly improve the migration function of LPS-stimulated trophoblast cells.Previous studies have demonstrated that impaired trophoblast cell function impeded spiral artery transformation, which may result in pre-eclampsia (6,7).Accordingly, DCA might have a therapeutic potential to prevent pregnancy complications with trophoblast disorder, such as pre-eclampsia, which remains to be validated in future studies.
Recently, it has been shown that DCA exhibits an antiinflammatory effect (15)(16)(17).DCA was found to alleviate the development of arthritis in female mice in an estrogendependent manner and affect macrophage migration function and inflammatory responses by inhibiting glycolytic reprogramming (15,16).Moreover, there is growing evidence that TLR4 has a critical effect during the activation of innate immune responses as a result of its pattern recognition ability (21).Several models and cell lines have been shown to respond to LPS by coordinating the expression of cytokines and other immune-related genes through TLR4 activation (19)(20)22).In the present work, it was demonstrated that DCA obviously inhibited LPSinduced overexpression of TNF-α, TLR4, IL-6, and IL-1β in HTR-8/SVneo cells.Moreover, we found that treatment with TLR4 inhibitors (CLI095 or MD2-TLR-4-IN-1) reduced overexpression of pro-inflammatory cytokines by LPS, and a synergistic effect was found between TLR4 inhibitors and DCA in HTR-8/SVneo cells (42)(43)(44)(45).From this, we speculated that the TLR4 signaling complex may be engaged in the mechanism by the DCA to inhibit LPSinduced inflammation in HTR-8/SVneo cells.
After TLR signaling complex activation at the plasma membrane, overexpression of pro-inflammatory mediators by transcription factors is triggered by the activation of a cascade of intracellular proteins (22).As the prototypical transcription factor, NF-κB has a pivotal role in the innate immune response.The expression level of NF-κB was reported to be obviously elevated in preeclamptic placentas in comparison with normal placentas (46).NF-κB can increase the expression of a diverse range of enzymes and pro-inflammatory cytokines by interacting with DNA and regulating the transcription of target genes, only if NF-κB is activated and translocated into the nucleus (21,23,(35)(36)(37).In this study, we found that DCA can markedly depress the phosphorylation of NF-κB (p65) and the translocation of p65 into the nucleus in HTR-8/SVneo cells stimulated by LPS.This finding suggests that DCA may regulate the LPSinduced inflammatory response in HTR-8/SVneo cells via the NF-κB pathway.

Conclusion
It was first shown that DCA improved trophoblast cell migration function by suppressing LPS-induced inflammation, at least partially via TLR4/NF-κB signaling pathway.(Figure 7).This result indicates that DCA might be a potential therapeutic candidate for human pregnancyrelated complications associated with trophoblast disorder.